Genetic and metabolic drivers of membrane remodeling in Clostridium thermocellum under alcohol stress

ABSTRACT Clostridium thermocellum is a leading candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels due to its native cellulolytic capabilities. Beyond ethanol, C. thermocellum is being developed as a platform for producing higher-chain alcohols such as isobutanol and n -butanol. However, its physiological adaptations to alcohol stress remain poorly understood. Here, we investigate how C. thermocellum remodels its membrane lipid composition in response to exogenous ethanol, n -butanol, isobutanol, and butyrate. Exposure to linear alcohols such as n -butanol or to organic acids like butyrate increased the proportion of straight-chain fatty acids in the membrane at the expense of branched-chain species, whereas exposure to the branched alcohol isobutanol produced the opposite effect. Isotope tracer experiments demonstrated that C. thermocellum directly incorporates the carbon backbones of exogenous alcohols and acids into fatty acids, providing a mechanistic basis for these contrasting shifts. We show that the bifunctional aldehyde/alcohol dehydrogenase AdhE is essential for the assimilation of exogenous alcohols into fatty acids, acting through its oxidative activity by first oxidizing alcohols to aldehydes and then converting them to acyl-CoA intermediates. Deletion of the pyruvate:ferredoxin oxidoreductase isozyme pfor4 abolished branched-chain fatty acid synthesis, but supplementation with isobutanol restored production, indicating that Pfor4 substitutes for the canonical branched-chain α-keto acid dehydrogenase complex. These findings reveal two distinct routes for branched-chain fatty acid production in C. thermocellum : a Pfor4-dependent pathway from α-keto acid intermediates derived from amino acid synthesis, and an AdhE-dependent salvage pathway that assimilates exogenous branched-chain alcohols. IMPORTANCE This study identifies key mechanisms of Clostridium thermocellum membrane remodeling under alcohol stress, showing that AdhE mediates incorporation of exogenous alcohols into fatty acids, while Pfor4 drives branched-chain fatty acid synthesis in the absence of the canonical Bkd complex. These findings highlight actionable targets for metabolic engineering to enhance solvent tolerance and improve the robustness and productivity of C. thermocellum as a biofuel-producing platform.